The smallpox virus, variola, has been responsible for the deaths of hundreds of millions of people worldwide. Although smallpox was eradicated from the globe in 1980 following a valiant immunization campaign, existing viral stocks may fall into the hands of those seeking to employ variola as a biological weapon. This calls for the development of safe antiviral therapeutics that will protect unvaccinated individuals for the time required to amount an immune response to the vaccine and individuals for whom the vaccine is contraindicated. Currently there are two therapeutics, brincidofovir and tecovirimat, which are in advanced stages of development against smallpox. However, drug-resistant brincidofovir and tecovirimat poxvirus variants arise during infection, and site-directed alterations of the variola genome could be readily constructed to deliberately engineer poxvirus mutants that are resistant to both drugs. Therefore, it is important to develop new therapeutics that recognize different poxvirus targets. Combinations of new and existing therapeutics will serve to circumvent both the natural and intentional generation of drug resistant variola. We have now discovered a platform technology involving small molecules, exemplified by 24a, that inhibit infection by vaccinia virus, the prototypic poxvirus and causative agent of smallpox. Early lead 24a acts via a novel mechanism in which it binds and destabilizes the vaccinia processivity factor D4, which is required by the D4/A20 processivity heterodimer for tethering the viral polymerase to the DNA template to enable processive or extended strand synthesis. Importantly, in the case of viral processivity factors, there are no cellular homologues, making them excellent drug targets. Moreover, our evidence indicates that early lead 24a may be broad-spectrum for all poxviruses, but not for other DNA viruses. Our goal is to (1) employ medicinal chemistry to optimize ADMET/PK and other properties while retaining/improving antiviral potency and creating new composition-of-matter intellectual property, (2) strategically mutate D4 at structurally defined residues to elucidate its binding mechanism, which will also help further guide medicinal chemistry, (3) examine the Broad Spectrum potential of our top optimized lead compounds for inhibition of several other poxviruses, including monkey pox, another bio- threat, and (4) evaluate the final top optimized compounds for their ability to protec mice against lethal poxvirus challenge. By the end of this SBIR Phase II period of study, our goal is to have identified one or more preclinical drug candidates with validated antiviral potency and safety, ADME/PK, and other required pharmaceutical criteria prior to conducting the pre-clinical development under the Animal Rule, required prior to filing an Investigational New Drug (IND) application with the U.S. FDA, as smallpox countermeasures that may also provide broad spectrum antiviral activity against other poxviruses.